In developing a binding assay, it is important that the scientist develop one that has a high level of sensitivity, precision and specificity, can eliminate interfering substances, and is convenient. The discussion herein may emphasize the immunochemical-type assay, but it should be recognized that the descriptions are also applicable to gene probe and other types of binding assays.
Sensitivity means the minimal detectable dose, namely the smallest mass of analyte that generates a statistically significant change in the signal generated by the assay vs. that obtained in the absence of analyte. There is a need to increase sensitivity of binding assays (i.e., detect smaller amounts of analyte), because in many situations the analytes, whether they are hormones, drugs, microorganisms, toxins, pollutants or genetic materials, exert their effects at low concentrations.
Furthermore, high sensitivity allows the use of small sample size, which can help to reduce "sample matrix" interferences. In addition, higher sensitivity allows measuring low analyte concentrations with a higher precision.
In discussing sensitivity, immunochemists have often distinguished between competitive assays and non-competitive assays. In a competitive assay, the signal which is measured is that emanating from the specific binder that does not bind analyte. For example, in some competitive assays, the labeled antibody is incubated with a sample containing analyte and a solid phase-immobilized analyte derivative. The labeled antibody that did not bind analyte binds the solid phase, and the signal emanating from the solid phase-bound labeled antibody is measured. In other types of competitive assays, unlabeled antibody is incubated with a sample containing an analyte and a labeled analyte derivative (or analyte mimic). The labeled analyte derivative binds those antibody binding sites that did not bind analyte. By measuring the signal coming from the labeled analyte derivative that bound the antibody, the assays actually obtains an estimate of the concentration of antibody sites that did not bind analyte. Thus, in both types of competitive assays, one measures signal associated with the fraction of specific binder sites that did not bind analyte. The signal generated from a competitive assay decreases as the analyte concentration increases. Since small levels of analyte correspond to large signals, small changes in low concentrations of analyte lead to small differences between large numbers, which are hard to measure accurately.
A second type of binding assay is the non-competitive type. In this assay, a labeled specific binder, for example a labeled antibody, is incubated with the sample and binds a portion of the analyte. In one variation (type A) of noncompetitive assay, a solid-phase immobilized unlabeled specific binder is added, simultaneously or in sequence, to bind another epitope on the analyte, in which case it is called a "sandwich" assay. For example, the immobilized molecule might be an antibody against a second epitope on the analyte, and the analyte might form a ternary complex with the labeled antibody and an immobilized unlabeled antibody. The solid phase is then washed and the signal measured is the signal that comes from the ternary complex containing the analyte. In this case the signal increases with increasing analyte concentration. Another variation of the non-competitive immunoassay (type B) was invented by L. E. M. Miles and C. N. Hales, Nature 219:186, 1968. In this type of assay the labeled antibody is first incubated with the analyte to form an immune complex, and then the mixture is contacted with a solid phase. This solid phase has an analyte derivative (or mimic) in large excess, which causes the unreacted labeled antibody to bind to it. The solid phase is then separated from the liquid phase and a portion of the liquid phase is taken for signal measurement. The difference from the competitive type of assay is that one does not measure the signal associated with the solid phase, namely the labeled binder that did not bind analyte. What one measures, instead, is the signal associated with the labeled binder that bound analyte and consequently did not bind the immobilized binder, thus remaining in the liquid phase. Improved versions of the non-competitive type B immunoassay include those invented by Baier et al. U.S Pat. No. 4,670,383, 1987 and Piran et al. U.S. Pat. No. 5,445,936, 1995.
Type A of the non-competitive assay has the potential for the highest sensitivity. Jackson and Ekins (T. M. Jackson and Ekins, R. P., Journal of Immunological Methods, 87:13, 1986) showed by mathematical analysis that when the specific activity of the label is not limiting, the sensitivity of type A is higher than that of the competitive assay. Empirical data supports the conclusion that type A of immunoassays is more sensitive than the competitive type of immunoassays: several immunoassays, such as thyroid stimulating hormone, have sensitivity of several million molecules per assay cuvette; in contrast, the most sensitive competitive immunoassays, such as those of digoxin and triiodothyronine, have sensitivities of several billion molecules per assay cuvette. There is a need to improve the sensitivities of non-competitive assays even further, and one way of achieving this improvement is via reduction of "nonspecific binding" (NSB), namely, the nonspecific adsorption of the labeled specific binder to the solid phase and the reaction vessel. Another way of increasing sensitivity according to Jackson and Ekins is to reduce the variability of the non-specific binding (NSB).
Interfering Factors
Often the sample to be analyzed in an immunoassay is delivered in an environment that includes interfering factors. For example, a serum sample not only contains the analyte of interest, but also many components that could interfere with the immunoassay. These interfering factor include not only well-defined and predictable molecules, present in higher-than-normal concentrations. However, they also can include materials which are not well-defined. Some immunochemical assay techniques include steps that isolate the analyte from the interfering substances. For example, the analyte can be reacted with an antibody which is connected to a solid phase. The solid phase can then be separated from the other components in its environment and incubated with a labeled specific binder, thus minimizing the contact of interfering factors with the labeled specific binder. However, the separation step in immunoassays is more often designed to only separate the bound portion of the labeled specific binder from the unbound portion. Although a small amount of the interferring substances may be eliminated via the traditional separation step, other means for eliminating the balance of the interferences from sample matrix are needed.
The separation step referred to above can be accomplished in one of many ways. For example: an assays can use non-magnetic particles as the solid phase using either centrifugation filtration as the method of separation, or magnetic particles as the solid phase, in which case the separation is accomplished by the application of a magnetic field. Other effective means of separation involve various chromatographies, electrophoreses, and the use of extended surfaces, such as microtiter plates, large beads, fibers and others. The separation step can be done manually or by an automated or non-automated instrument; in either case, however, the solid phase is separated and washed, the liquid phases are discarded, and the solid phase-associated signal is the one being measured.
Many substances interfere with the assays despite the wash steps. For example, cross-reactants share structural similarities with the analyte and also bind the labeled or unlabeled specific binder. When a cross-reactant binds the labeled specific binder the assay result is falsely elevated. When sufficiently high concentration of a cross-reactant binds the unlabeled specific binder and saturates it, a false result is obtained.
Occasionally the analyte itself is present in an extremely high concentration, causing an interference in two-site (sandwich) noncompetitive assays known as "high dose hook effect." This interference manifests itself as a falsely low signal, which makes a very high dose to be confused with a low dose. Other interfering factors are heterophilic antibodies, human anti-mouse IgG antibodies, human antibodies to gamma globulin of other animal species, rheumatoid factors and other macromolecules present in the sample can bind antibodies and can either form a bridge between the labeled and unlabeled antibodies or inhibit their desired binding activity, leading in each case to false results. Similar interfering factors are present in other binding assays, such as gene probe assays. The effect of these interferences is usually to provide inaccurate result; however, in the low analyte concentration region interfering factors can increase the variability of the "zero dose signal" and thus reduce sensitivity. Therefore, correction for interfering factors can increase sensitivity of the assay.